Programmable, continuous flow analyzer

ABSTRACT

Method and device for continuous flow-through analysis with a non-segmented laminar carrier flow, to which reagents are added at programmable intervals. The programming can be done by switching in various loops with valves, interchangeable connections or by composing the apparatus of different prefabricated modules to produce the desired reaction conditions.

This is a continuation, of application Ser. No. 878,265, filed Feb. 16,1978 now U.S. Pat. No. 4,224,033.

The present invention relates to a programmable, continuous flowanalyzer with a continuous, unobstructed laminar carrier flow which isnot segmented by air bubbles.

Methods of rapid, exact, chemical analysis of separate samples are veryimportant because of the increasing need for analysis in chemical andbiochemical investigations of environmental problems, food problems,clinical studies, etc. The development of methods of analysis andimproving the speed of analysis are dependent on the possiblities ofproducing an apparatus which performs rapid and exact analyses and whichhas a broad range of use.

In principal, there are two different paths which have been followed inthe development of apparatus for high-speed analysis. One of theminvolves the use of an apparatus which places each sample and theappropriate reagent in an individual container, in a manner similar tothat of a laboratory technician in manual analysis. Even if thisprocedure has many advantages, the required apparatus is quitecomplicated.

The other path involves using a continuous reaction flow and providesquite rapid analysis of many different substances with relatively simpleapparatus.

The greatest problem in continuous flow reaction systems has been tomaintain the integrity of the samples. One of the pioneers in the field,Skeggs, Amer. J. Clin. Path, 28, 311-322, (1957), has developed a systemwith air bubbles between the different samples. The majority ofmechanized colorimetric analyzers are now based on this system ofsegmentation of the reaction flow with air bubbles.

Even if the system with air segmentation is excellent at low speeds ofanalysis, 10-30 samples/h, it is difficult to obtain reference readingsat higher speeds, 60-120 samples/h, and poor analytical accuracy isobtained.

The basic principles and characteristics of automatic analyzers aregiven in more detail in our U.S. Pat. No. 4,177,677, which is especiallydirected to supplying samples to a continuous carrier flow.

In the apparatus produced according to our earlier abovementioned patentapplication, the problems with dispersion of the samples has beensolved, so that different analyses produce comparable results. However,a remaining problem which is closely associated with the dispersionproblem is the supplying of two or more converging flows to a reactionsystem, and at the same time making optimal reaction conditions possiblein connection with the addition of the reagents.

These problems are solved by the present invention, which makes itpossible to add two or more different flows so that they becomeconfluent and to vary the length of a main conduit during operation byswitching in or switching out different conduit segments arbitrarily oraccording to need.

The invention will be described in more detail below in connection withexamples and with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings show in

FIG. 1 a flow chart for the apparatus according to the invention,

FIG. 2 the graph of the dispersion of a sample zone,

FIG. 3 a graph showing pH measurement at low dispersion,

FIG. 4 a flow chart for another apparatus according to the invention,

FIG. 5 graph of acid titration with high dispersion,

FIG. 6 nitrogen determination at medium dispersion, and in

FIG. 7 a schematic drawing of the apparatus according to the inventionconstructed with modules.

FIG. 1 shows a schematic flow chart for a apparatus according to theinvention in a simplified form. The reference numerals 1-10 represent anumber of input or output conduits to valves or cocks 12-19, which forthe sake of simplicity are shown as three-way valves. A number of loopsA-D of different lengths are connected to these valves together withapparatus.

Thus loop A consists of a conduit 20 from valve 12 to a coil 21 and fromthere to a sample injection device 22 with a shunting circuit 23. Asdesired, the valve 12 can join the loop A to conduit 1 or 2 or with bothof these conduits at the same time. The sample injection device isdescribed in detail in our Swedish Patent Application No. 7610110-4.

From the sample injection the conduit 24 continues to a back mix 25coupled between three-way valves 26 and 27 and with a shunting circuit28 between the valves enabling the back mix to be switched in and out asdesired. From valve 27 the conduit 29 continues to valve 13 to becoupled there either to conduit 3 or via a connecting conduit 30 tovalve 14 or is entirely closed off.

Valve 14 connects conduit 30 with conduit 4 or with loop B via conduit31 or with both of these conduits. The conduit 31 goes to the coil 32,and from there conduit 33 continues to valve 15. In the same manner,loop C, 35,36,37, is connected between valves 16 and 17 and loop D,39,40,41 between valves 18 and 19.

In a similar manner conduits 2 and 3 or valves 12 and 13 can be joinedto one another via conduit 42 and valves 43 and 44, i.e. by-passing loopA. Conduits 4 and 5 can be coupled together with the connecting conduit45 and valves 46 and 47; conduits 6 and 7 with connecting conduit 48 andvalves 49 and 50; and conduits 8 and 9 with connecting conduit 51 andvalves 52 and 53.

Thus in the apparatus shown a carrier flow can be supplied through, forexample, conduit 1, possibly mixing it with a supplementary carrier flowvia conduit 2. It then passes the coil 21 to even out the flow, is mixedwith a sample solution in the sample injection device 22, is titrated inthe back mix 5, is led to the outlet via conduit 3 or continues throughone or more of the loops B,C and D and is thereby mixed with differentreagents via one or more of the conduits 3-9 and is finally led to ameasuring instrument, a colorimeter, a measuring electrode, etc. viaconduit 10.

Other apparatus, instruments or loops can likewise be coupled in betweentwo of the conduits 3-9 or the flowing solution can be taken out throughone of the conduits as desired for special purposes. The flow can alsobe directed in the other direction adding the reagent via one or more ofthe conduits 3-10 and having the outlet for measurement through conduit1 or 2.

The number of possible variations is quite large, and the examples givenabove are in no way exhaustive.

The procedure in several different analyses will be described in thefollowing examples, in which loops A,B,C and D had lengths of 80, 20, 60and 80 cm respectively and are kept at a temperature of 38° C. bythermostated baths. All of the conduits had a diameter of 0.5 mm and theconnecting conduits 42,45,48 and 51 were switched out with therespective valves.

EXAMPLE 1

Chloride determination.

Programming.

Conduit 1 closed.

Conduit 2 addition of 1.5 ml/min. of a solution containing per liter 15%ethanol in water: 0.626 g mercury(II)thiocyanate, 30 g iron(III)nitrateand 4.7 g nitric acid, 26,28,27 back mix switched out.

Conduits 3 and 4 closed.

Conduit 5 coupled to flow-through cell, 10 mm, volume 18 μl(microliters), for measurement at 480 nm.

Conduits 6-10 closed.

With a sample volume of 30 μl a usable measuring range of 0.5-20 ppm Clwas obtained, and with a sample volume of 10 μl a usable measuring rangeof 10-50 ppm Cl was obtained.

EXAMPLE 2

Ammonia determination.

Programming.

Conduit 1 closed.

Conduit 2 addition of 1 ml water/min. 26,28,27 backmix switched out.

Conduit 3 addition of 1 ml/min. of a solution containing per liter 33%ethanol in water: 50 g phenol and 120 g NaOH.

Conduit 4 closed.

Conduit 5 addition of 1 ml/min. of a solution of 4% Cl₂ in water andcontaining per liter 20 g NaOH and 20 g borax.

Conduit 6 closed and conduit 7 coupled to a flow-through cell, 10 mmlong, volume 18 μl for measurement at 620 nm.

Conduits 8-10 closed.

Sample volume 30 μl and usable measuring range 1.0-25 ppm NH₃.

EXAMPLE 3

Determination of pCa in serum.

Programming.

Conduit 1 closed, conduit 2, 3.0 ml/min 0.14 N NaCl and 26,28,27 backmix switched out. Conduit 3 coupled to flow-through cell with calciumelectrode and conduits 4-10 closed.

Sample volume 30 μl and usable measuring range 5×10⁻⁴ -5×10⁻³ M Ca²⁺(pCa 2.7-3.7).

For the sake of illustration, in these examples the outlet formeasurements was made at different points, but the usual case is that ameasuring instrument or a measuring cell is permanently coupled in at 10and that the fluid flow for measurement is conducted there by makingconnections between the existing conduits. For the sake of clarity, inFIG. 1 the valves are shown as three-way valves, for example valves 46and 14 in conduit 4. In practice a single valve is used instead of thetwo valves and it can be electrically controlled so that the entireprogramming is done from a clearly arranged control panel where the flowpaths are set and controlled directly. Likewise, the valves orinterchangeable connections can be made with several functions so thatvarious loops are coupled in parallel between two valves and the loopdesired at that particular time can be switched in by the programming onthe control panel.

The dispersion of the sample plug in the carrier solution is of vitalinterest in analysis according to the invention, and the analysisapparatus according to the invention can be programmed to producedifferent degrees of dispersion. At low dispersion of the sample plugthe dilution at the front and rear edges of the plug will be minimal andthe gradient curve will thus be quite steep, while a high dispersionproduces a gentle gradient curve. The degree of dispersion is determinedby the velocity of the flow, the length and inner diameter of the loops,and different effects in analysis are obtained by programming fordifferent degrees of dispersion.

The dispersion of the sample zone in a tube with laminar flow followsthe formula ##EQU1## in which C designates the sample concentration, Lis the length of the reactor loop and N and 1_(s) are constants of thesystem.

The formation of the colored product of a chemical reaction is often acase of first order kinetics where for a certain pumping speed thefollowing relationship is valid:

    C/C.sub.max =1-e.sup.-kL                                   (2)

where k is a constant.

Thus, in the simple case where N=, Equation (1) is depicted by curve (1)in FIG. 2, while Equation (2) is represented by curve (2) in the samefigure. The resulting curve (3) is then a characteristic for each flowsystem where it can be obtained by injecting the same sample and varyingeither L or the pumping speed.

The number N is, in fact, the number of theoretical mixing chambers. Inthe automatic titration procedure special advantage is taken of the casewhere N=L and k is very large (instant reaction); here a relativelylarge mixing chamber serves as the chemical reactor. We will return tothe theories behind this in connection with the following examples.

These examples were carried out with an apparatus according to FIG. 3,in which the corresponding numerals and letters have the same meaningsas in FIG. 1 and with the following dimensions for the loops: A 60 cm,0.5 mm, B 20 cm, 0.5 mm, C 30 cm, 0.75 mm, D 30 cm, 0.75 mm and E 60 cm,0.75 mm. 54 designates a flow-through cell of the type described inSwedish Patent Application No. 7610221-9 and is coupled in between 26and 27.

EXAMPLE 4

Low dispersion.

Measurement of pH with glass electrode in flow-through cell.

Measuring range ca 5 pH units, sample volume 30 μl and analysis rate 475samples/h.

Programming.

Conduit 1 closed, conduit 2 reagent inlet, 3.0 ml/min. and conduit 3outlet, 4.0 ml/min. Conduits 4-8 closed, 26,27 coupled to a flow-throughcell for measurement with glass electrode. By using a special electrodedesign according to our Swedish Patent Application No. 761221-9 with thesolution flowing over the surface of the electrode, the outflowingamount will be greater than the inflowing.

As a reagent a buffer solution with pH 6.70 was used with a composition1.0×10⁻³ M Na₂ HPO₄, 1.0×10⁻³ M NaH₂ PO₄, 1.0×10⁻¹ M NaCl. Thecomposition of the reagent solution is suitably selected so that its pHlies in the middle of the pH values of the samples.

Measurements on 9 standard buffer solutions in the pH range 4.6-9.2 areshown in FIG. 3 with 4-6 different measurements of each solution. Theaccuracy can be seen by the agreement of the height of the peaks.

EXAMPLE 5

High sample dispersion. A strong acid is titrated with a strong base.

Programming.

Conduit 1 closed, conduit 2 addition of 1.3 ml/min. 5×10⁻³ M NaOHcontaining per 500 ml;: 1 ml indicator consisting of 0.4 g BromothymolBlue, 25 ml 96% ethanol and distilled water to 100 ml. Conduit 3 iscoupled to a flow-through cell, 10 mm, 18 μl for measurement at 620 nm.Conduits 4-10 closed, 26-27 open to the mixing chamber at 1 ml andcontaining a magnetic stirrer, so that an effective agitation in themixing chamber is obtained when placed on a magnetic stirring table.

The sample volume was 200 μl, and standard solutions of dilutedhydrochloric acid were produced by successive dilution of a basesolution with distilled water. The measurements were done at roomtemperature, and the usuable measurement range was 2×10⁻² M-5×10⁻¹ M orthe equivalent amount of another strong acid.

The quantitative determination of the acid concentration by titration ofthe acid HCl with the base NaOH is done by the acid samples beinginjected in a flow-through system according to FIG. 4, in which adilution gradient is created in the mixing chamber, cf. FIG. 2, curve(1). As a reagent flow a 1.0×10⁻³ M NaOH solution was used containingthe indicator Bromothymol Blue. Through injection of an acid sample ofsufficiently high concentration, the indicator in the mixing chamberchanges to yellow, but the acid sample is then gradually diluted by thecontinuous pumping in of carrier solution and when its concentration isless that that of the carrier flow, a second color change occurs to theblue basic color, i.e. the change of color marks the point ofequivalence, and the time between the two changes in color discloses theconcentration of the acid. The monitoring for the actual color change iscontinuous via a flow-through cell mounted in a spectrophotometer andset to a wavelength of 620 nm.

Provided that the injected sample is added to the mixing chamber as apulse and then assumed to be mixed homogeneously with the carrier flowbefore it begins to exit from the chamber, it can be shownmathematically that the dilution gradient follows the equation

    C.sub.t =C.sub.o e.sup.- vt/V                              (3)

where C_(t) is the concentration of the acid at time t, C_(o) theconcentration at time o, i.e. when the entire sample is still inside thechamber, v is the speed of the pump in ml/min, t the time in minutes andV the volume of the mixing chamber in ml.

After taking logarithms and converting to base-10 logarithms, theequation (3) can also be written

    log C.sub.t =log C.sub.o -tv/(Vln 10)                      (4)

or

    t=(V/vln 10 log C.sub.o -(V/v) 1n 10 log C                 (5)

When an acid pulse is injected in the basic carrier flow, and providedthat the acid-base reaction occurs instantaneously, the point ofequivalence is reached after a time t_(eq) at which log C_(HCl) =logC_(NaOH) =log C, i.e.

    t.sub.eq =(V/v) l n 10 log C.sub.o -(V/v) ln 10 log C.sub.NaOH (6)

that is to say the last term is a constant. A graph mapping of t_(eq)against log C_(o), based on a series of acid standards, produces astraight line with the slope V/v ln 10, and from this calibration curveit is possible by measuring t_(eq) in a given sample to determine itsinitial concentration C'_(o).

In Equation (6) C_(o) is, as was stated earlier, the concentration inthe mixing chamber, but when the sample volume is held constant, in thiscase 200 μl, the diagram can be read instead C'_(o) =initialconcentration of sample.

Extrapolated to t_(eq) =0, the equation can be reduced to log C_(o) =logC_(NaOH), i.e. the value of log C_(NaOH) is directly reflected in thesensitivity limit of the process.

FIG. 5 shows the graphed results. The time t has been marked for eachsample, and it is proportional to the width of the peak in mm and to thelogarithm of the concentration in the sample. The color of the solutionis blue in the lower portion of the curve and yellow in the upperportion.

EXAMPLE 6

High sample dispersion.

A strong acid is titrated with a strong base.

A controlled concentration gradient of the injected acid sample isobtained and the sample is then mixed continuously with a solution of abase containing an acid-base indicator whose color can be measuredcontinuously. The range of measurement is about 0.8 of the concentrationdecade. The quantative evaluation is made as previously by measuring thehalf-width of the registered signal, which is proportional to thelogarithm of the concentration. The relative position of the range ofmeasurement is a function of the molarity of the reagent solution, sothat for example a 10⁻³ M NaOH solution gives a range of measurement ofabout 4×10⁻³ -2×10⁻² M H⁺.

The sample volume was 30 μl and the greatest measuring speed was 60samples/h.

Programming for the same apparatus as in Example 4.

Conduit 1 closed, conduit 2 distilled water 1.66 ml/min. and conduit 3closed. Conduit 4 reagent addition 1.66 ml/min., conduits 5-6 closed andconduit 7 open to the flow-through cell. Conduit 8 closed and 26-27 opento a gradient tube with length 25 cm and inner diameter 1.70 mm.

Reagent solution: Diluted sodium chloride containing per 500 ml: 1 mlindicator consisting of 0.4 g Bromothymol Blue, 25 ml 96% ethanol anddistilled water to 100 ml.

Standard solutions with diluted hydrochloric acid were prepared bysuccessive dilution of a main solution with distilled water. Themeasurements were done at 620 nm and at room temperature.

According to the flow chart on FIG. 4 distilled water enters through 2,passes through the loop A to the sample injection 22, from there to 26and through the gradient tube to 27, through the loop E to 4 where thereagent solution is added and then through C and D and out through 7 tothe measuring cell.

Between the low and the high degrees of dispersion there is a largerange of use with medium dispersion of the sample plug.

EXAMPLE 7

Medium-dispersion.

Measurement of Kjedahl nitrogen.

Ammonia is oxidized with hypochlorite to chloramine, which then reactswith phenol to Indolphenol Blue whose color intensity is measuredcolorimetrically. The range of measurement is 1-3.5% N, where %N refersto the percentage of dry vegetable matter fused according to the usualKjedahl method with 300 mg vegetable matter in a final volume of 50 mlwith the acidity of 1.0 M sulfuric acid. It is important that air beremoved from the alkaline reagent solution before use, for example in anErlenmeyer flask with a water jet pump. Furthermore the flasks should beprotected from the atmosphere to prevent absorption of carbon dioxide.

The sample volume is 30 μl, the maximum speed is 90 samples per hour andthe highest sensitivity is 0.75% N.

Programming.

Conduit 1 closed, conduit 2 addition of reagent 1 at 1.66 ml/min, andconduits 3-5 closed. Conduit 6 addition of reagent 2 at 1.23 ml/min.,conduit 7 closed, conduit 8 open to the flow-through cell, 26,27 closed.

Reagent 1 was made up of 25.0 g phenol, 60.0 g sodium hydroxide, 150 ml96% ethanol and distilled water to 1.0 liter. Reagent 2 consisted of20.0 g sodium hydroxide, 20.0 g borax, 800 ml bleaching solutioncontaining 5% active chlorine and distilled water to 1.0 liter. Standardsolutions were prepared by diluting an ammonium sulphate solution with8% N with diluted sulfuric acid.

The measurements were taken at a temperature of 34° C. at 620 nm in ameasuring cell with a 10 mm optical path and volume 18 μl. The graph ofthe results is given in FIG. 6.

As is evident from the above, the analysis apparatus according to theinvention has a very wide range of use. The sample amounts required arequite small, in some cases less than 10 μl, even though as a rule samplequantitites of 20-30 μl are used. However, there is no obstacle to theuse of larger sample quantities of 60-70 μl and up to 100 μl or evenseveral hundred μl. The dimensions of the loops and the conduits must bedependent on the sample quantities used so that the flow rate will besufficiently low to prevent undesirable turbalence, i.e. a sufficientlylow Reynolds number to maintain the desired degree of dispersion in eachcase. Reynolds numbers below 500 are usually suitable, and numbers inthe range 10-150 have been used with success. In certain cases aReynolds number as low as 2 has been used. When analyzing organicsamples, the viscosity can be appreciably greater than for ordinarysimple inorangic acids or bases, and the flow conditions must then bechanged, of course.

The diameter of the conduits is also of importance for the flow and forsmall samples we have found a diameter of 0.25 mm to be about right eventhough we have used smaller diameters, 0.10 or the like. But with largersample quantities a larger diameter must be used, such as 0.5 mm, 1.0 mmand even greater. In the gradient tube according to Example 6, adiameter of 1.7 mm is used and even larger diameters can be used.

The flexibility of the apparatus can, as has already been mentioned, bemade quite high by coupling various loops in parallel between the valvesand the desired loop can be switched in or out by a simple valveoperation or by replacing an interchangeable connection. We have,however, also used with success a construction in which loops cast in aplastic block or coupled to a plastic block are inserted in a socket,thus connecting them sealingly into the system. In this way simplethree-way valves at positions 12-19 can make the valves 43,44,46,67 etc.unnecessary, while preserving the same coupling possibilities, but withunlimited flexibility. A coupling with the more extensive valve systemis suitable when a limited number of different types of analyses are tobe done, while for research purposes or for the development of newmethods, the latter method with insertable loops, provides unlimitedvariations at very low cost.

When only a few types of standard analyses are required, it can beadvantageous to replace all of the valves with programmed gauge blocksor cassettes, of plastic for example, which directly block all of theundesired connections, and which connect those points which are to beconnected for the analysis in question by interchangeable connections.

FIG. 7 shows an embodiment with such a cassette inserted for ammoniadetermination according to Example 2. This cassette fulfills the samefunctions as the valves shown in FIG. 1, and has the advantage ofpreventing misprogramming. The seal at the connections is in itselfquite sufficient, with ordinary slide fitting between the plasticsurfaces, but the cassettes can also be provided with cut grooves andinserted gaskets around the openings.

Likewise, an arbitray number of valves can be arranged in the cassettes.Thus all of the valve functions shown in FIG. 1 can be housed in acassette even if valves are entirely avoided in most cases when usingcassettes for programming, or possibly with one or two valves.

What we claim is:
 1. A method of preparing a sample for treatment inwhich a continuous flow of liquid carrier receives sample portions, saidmethod comprising: passing said carrier through a conduit in a mannersuch that flow of said carrier is laminar, unsegmented and continuous,introducing sample portions into said carrier, controlling dispersion ofsaid sample portion in said carrier by varying at least one of thefollowing: the volume of said sample portion, the flow velocity of saidcarrier, or the dimensions of said conduit conducting said sample andsaid carrier.
 2. The method of claim 1 wherein dispersion of said sampleportion and said carrier is controlled by varying the length of theconduit through which said sample and said carrier is passed.
 3. Themethod of claim 1 wherein dispersion of said sample portion and saidcarrier is controlled by varying the diameter of said conduit.
 4. Themethod of claim 1 wherein dispersion of said sample portion and saidcarrier is controlled by varying the volume of said sample.
 5. A methodof treating a sample in which a continuous flow of a liquid carrierreceives sample portions, said method comprising: passing said carrierthrough a conduit in a manner such that flow of said carrier is laminar,unsegmented and continuous, controlling dispersion of said sampleportions in said carrier by varying at least one of the following: thevolume of said sample portion, the flow velocity of said carrier, thedimensions of said conduit conducting said sample and said carrier; andintroducing a treatment material into said sample as it is carriedthrough said conduit.
 6. The method of claim 5 wherein the treatmentmaterial is a reagent which chemically reacts with said sample.
 7. Anapparatus for continuous flowthrough treatment of samples comprising:amain conduit for conducting a liquid carrier, said conduit beingdisposed to conduct said carrier in a manner such that the flow of saidcarrier is laminar, unsegmented and continuous, means for insertingsample plugs into said carrier at controlled intervals, and means forcontrolling the dispersion of said sample plugs in said carrier.
 8. Anapparatus for continuous flowthrough treatment of a sample comprising:amain conduit for conducting a liquid carrier, said conduit beingdisposed to conduct said carrier in a manner such that the flow of saidcarrier is laminar, unsegmented and continuous; means for insertingsample plugs into said carrier at controlled intervals; means forintroducing a treatment material into said carrier; and means forcontrolling the dispersion of said sample plugs in said carrier.
 9. Themethod of claim 1 wherein dispersion of said sample portion and saidcarrier is controlled by varying the flow velocity of said carrier.